While relatively rapid and convenient procedures for the purification of nucleic acid (such as DNA) from agarose have been developed, it remains a relatively difficult operation to extract nucleic acid directly from more complex starting samples such as cells and cell lysates. On the whole, the procedures currently practiced to purify nucleic acid from nucleic acid-containing samples comprising cells or cell lysates remain time consuming and labor intensive.
One method proposed to minimize the laborious and time-consuming steps of the known method for isolating nucleic acid from these more complex sample is described in EP 0389063. The method disclosed in EP 0389063 involves mixing the cell sample (such as whole blood) with a chaotropic substance and a particulate nucleic acid binding solid phase comprising silica or a derivative thereof. It is known that, in the presence of a chaotropic substance, nucleic acid is released from cells and binds to silica-based nucleic acid binding solid phases. Subsequently, the mixture is centrifuged to pellet the solid phase with the nucleic acid bound thereto and the supernatant is discarded. The pelletized material is subjected to several washing stages with the chaotropic agent and organic solvents. Finally, the DNA is eluted from the solid phase in a low salt buffer.
The method described in EP 0389063 is disadvantageous in that it is a manually intensive, multi-step procedure. In view of the fact that the method involves a number of centrifugation and vessel transfer steps, this method is unsuitable for automation.
U.S. Pat. Nos. 5,187,083 and 5,234,824 each describe a method for rapidly obtaining substantially pure DNA from a biological sample containing cells. The methods involve gently lysing the membranes of the cells to yield a lysate containing genomic DNA in a high molecular weight form. The lysate is moved through a porous filter to trap selectively the high molecular weight DNA on the filter. The DNA is released from the filter using an aqueous solution.
The present invention aims to provide an improved method for isolating nucleic acid from a nucleic acid-containing sample, such as cells or cell lysate, which avoids the use of centrifugation steps and which avoids the requirement of upstream processing of the sample in order to render the nucleic acid amenable to binding to a solid phase.
Genotyping is the discipline of identifying an individual's genome in relation to disease specific alleles and/or mutations that occur as an effect of parental linkage. The rapid purification of human genomic DNA is an essential part of a genotyping process; the genomic DNA of an individual being the structural unit for the entire DNA sequence of every allele expressed.
Human genomic DNA cannot be directly sequenced. In order to carry out sequence analysis on regions of the chromosomes that may contain portions of mutation or disease specific sequences, selected portions are amplified, e.g., via PCR, and the amplified products are sequenced. The selected portions of the chromosomes that are amplified are dictated by the specific sequence of the primers used in the PCR amplification. The primer sets that are used in genotyping studies are commercially available and are representative for the chromosome under examination. If linkage studies identify that a disease-bearing sequence is on a particular chromosome, then many primer sets will be utilized across that chromosome in order to obtain genetic material for sequencing. The resultant PCR products may well represent the entire chromosome under examination. Due to the large length of chromosomes, many PCR reactions are carried out on the genomic DNA template from a single patient.
Human genomic DNA is purified by a variety of methods (Molecular Cloning, Sambrook et al. (1989)). Consequently, many commercial kit manufacturers provide products for such techniques, for example: AmpReady™ (Promega, Madison, Wis.), DNeasy™ (Qiagen, Valencia, Calif.), and Split Second™ (Roche Molecular Biochemicals, Indianapolis, Ind.). These products rely on the use of specialized matrices or buffer systems for the rapid isolation of the genomic DNA molecule.
More recently, microporous filter-based techniques have surfaced as tools for the purification of genomic DNA as well as a whole multitude of nucleic acids. The advantage of filter-based matrices are that they can be fashioned into many formats that include tubes, spin tubes, sheets, and microwell plates. Microporous filter membranes as purification support matrices have other advantages within the art. They provide a compact, easy to manipulate system allowing for the capture of the desired molecule and the removal of unwanted components in a fluid phase at higher throughput and faster processing times than possible with column chromatography. This is due to the fast diffusion rates possible on filter membranes.
Nucleic acid molecules have been captured on filter membranes, generally either through simple adsorption or through a chemical reaction between complementary reactive groups present on the filter membrane or on a filter-bound ligand resulting in the formation of a covalent bond between the ligand and the desired nucleic acid.
Porous filter membrane materials used for non-covalent nucleic acid immobilization have included materials such as nylon, nitrocellulose, hydrophobic polyvinylidinefluoride (PVDF), and glass microfiber. A number of methods and reagents have also been developed to also allow the direct coupling of nucleic acids onto solid supports, such as oligonucleotides and primers (eg. J. M. Coull et al., Tetrahedron Lett. vol. 27, page 3991; B. A. Conolly, Nucleic Acids Res., vol. 15, page 3131, 1987; B. A. Conolly and P. Rider, Nucleic Acids Res., vol. 12, page 4485, 1985; Yang et al., P.N.A.S. vol. 95: 5462-5467). UV cross-linking of DNA (Church et al., PNAS, vol. 81, page 1991, 1984), The Generation Capture Column Kit (Gentra Systems, Minneapolis, Minn.) and RNA (Khandjian et al., Anal. Biochem, vol. 159, pages 227, 1986) to nylon membranes have also been reported.
Many chemical methods have been utilized for the immobilization of molecules such as nucleic acids on filter membranes. For example, activated paper (TransBind™, Schleicher & Schuell Ltd., Keene, N.H.) carbodimidazole-activated hydrogel-coated PVDF membrane (Immobilin-IAV™, Millipore Corp., Bedford, Mass.), MAP paper (Amersham, Littlechalfont Bucks, Wis.), activated nylon (BioDyne™, Pall Corp., (Glen Cove, N.Y.), DVS- and cyanogen bromide-activated nitrocellulose. Membranes bound with specific ligands are also known such as the SAM2™ Biotin Capture Membrane (Promega) which binds biotinylated molecules based on their affinity to streptavidin or MAC affinity membrane system (protein A/G) (Amicon, Bedford, Mass.). Some of the disadvantages of covalent attachment of biomolecules onto activated membranes are:                a) Molecule immobilization is often slow, requiring 20-180 minutes for reaction completion.        b) High ligand and biomolecule concentration is needed for fast immobilization.        c) Constant agitation is needed during the immobilization process, which may result in biomolecule denaturation and deactivation.        d) Once the immobilization process is complete, often a blocking (capping) step is required to remove residual covalent binding capacity.        e) Covalently bound molecules can not be retrieved from the filter membrane.        
There is a need in various specific areas, such as forensics, for a nucleic acid immobilization media and procedure that exhibits the high specificity of covalent immobilization onto the filter membrane without the use of harsh chemical reactions and long incubation times, which can also be used at crime scenes, with blood sample archiving and other related uses. In particular there is a need for the capture and separation of nucleic acids from a mixture in a fluid phase onto a filter membrane matrix in forensics.
Of special interest is the ability to store or archive the bound nucleic acids on the filter membrane matrix for various uses. Alternatively, filters that permit elution of nucleic acids have uses in application requiring liquid formats. Embodiments of both types are found in the present invention.
More recently, glass microfiber, has been shown to specifically bind nucleic acids from a variety of nucleic acid containing sources very effectively (for example, see Itoh et al (1997) Simple and rapid preparation of plasmid template by filtration method using microtiter filter plates. NAR, vol. 25, No. 6: 1315-1316; Andersson, B. et al. (1996) Method for 96-well M13 DNA template preparations for large-scale sequencing. BioTechniques vol. 20: 1022-1027). Under the correct salt and buffering conditions, nucleic acids will bind to glass or silica with high specificity.
Based on U.S. Pat. Nos. 5,496,562, 5,756,126, and 5,807,527, it has been demonstrated that nucleic acids or genetic material can be immobilized to a cellulosic-based dry solid support or filter (FTA filter). The solid support described is conditioned with a chemical composition that is capable of carrying out several functions: (i) lyse intact cellular material upon contact, releasing genetic material, (ii) enable and allow for the conditions that facilitate genetic material immobilization to the solid support (probably by a combination of mechanical and chaotrophic), (iii) maintain the immobilized genetic material in a stable state without damage due to degradation, endonuclease activity, UV interference, and microbial attack, and (iv) maintain the genetic material as a support-bound molecule that is not removed from the solid support during any down stream processing (as demonstrated by Del Rio et al (1995) BioTechniques. vol. 20: 970-974).
The usefulness of the so called FTA cellulosic filter material described in U.S. Pat. Nos. 5,496,562, 5,756,126, and 5,807,527 has been illustrated for several nucleic acid techniques such as bacterial ribotyping (Rogers, C. & Burgoyne, L. (1997) Anal. Biochem. vol. 247: 223-227), detection of single base differences in viral and human DNA (Ibrahim et al. (1998) Anal. Chem. vol. 70: 2013-2017), DNA databasing (Ledray et al. (1997) J. Emergency Nursing. vol. 23, No. 2: 156-158), automated processing for STR electrophoresis (Belgrader, B. & Marino, M. (1996) L.R.A. vol. 9: 3-7, Belgrader et al. (1995) BioTechniques. vol. 19, No.3: 427-432), and oligonucleotide ligation assay for diagnostics (Baron et al. (1996) Nature Biotech. vol. 14: 1279-1282).
It has been shown that nucleic acid or genetic material applied to, and immobilized to, FTA filters cannot be simply removed, or eluted from the solid support once bound (Del Rio et al (1995) BioTechniques. vol. 20: 970-974). This is a major disadvantage for applications where several downstream processes are required from the same sample, such a STR profiling and genotyping.
FTA filter media has also been employed for DNA analysis and comparison in other ways. For example, cellular material has been applied to FTA filter media, and generally the cellular material, once applied, forms a spot on the FTA filter. From this spot, small punches can be taken; each small punch will have immobilized to it enough nucleic acid or genetic material to facilitate a single downstream process such as a PCR reaction. As the two primers administered to a PCR reaction are presented in solution, it is of no consequence that the cellular nucleic acid template is immobilized to the filter. All amplicon will be formed in solution. Amplicon can then be readily removed from the reaction by aspirating the liquid phase away from the FTA solid filter punch. Therefore, for multiple processing from a single sample, many punches have to be taken. Multiple punching is very time consuming, and as yet, has not lent itself to simplified automation.
A primary use for the present invention is the analysis of blood transfusion products for leukocytes or other contaminants. Each year in the United States about 14 million transfusions of blood or blood components takes place. There are three major blood products in transfusion medicine:                1. RED CELLS (RBC, typically about 340 ml contained in 1 unit of donor blood)—the remaining red cell mass after most of the plasma is removed.        2. PLATELETS (typically 300 ml/1 unit of donor blood) or platelet concentrates (PCs, typically further concentrated to about 50 ml/1 unit of donor blood)—one platelet concentrate (one unit of random donor platelets) is derived from one unit of donor blood.        3. FRESH FROZEN PLASMA (FFP, 225 ml/1 unit of donor blood)—One unit of FFP can raise coagulation factor levels by 8% and fibrinogen by 13 mg/dl in the average patient.        
Despite the increasing need for transfusions and the use of transfusion products, such use involves a number of risks. About 150,000 patients a year experience adverse reactions to such products. Such adverse reactions occur regardless the type of blood transfusion a patient receives. Ninety percent of adverse transfusion reactions are caused by donor leukocytes contained in the transfusion products.
Further problems stem from Human Leukocyte Antigen (HLA) alloimmunization, in which the recipient is sensitized to antibodies contained in the transfusion product which can react, for example, to the recipient's leukocytes (HLA sensitization).
Where the recipient suffers from a non-hemolytic febrile transfusion reaction, the patient most frequently experiences fever, chills, and nausea due to white blood components contained in the transfusion product, to which the patients has antibodies (usually anti-HLA).
Other serious risks of the use of transfusion products include transmission and/or reactivation of cytomegalovirus (CMV), occurrence of graft-versus-host disease (GVHD), and the risk of viral transmissions. (HIV, HCV transmission are the most feared complications of transfusion.)
Certain precautions have been adopted in order to reduce the likelihood and/or severity of adverse reactions to transfusion products. Leukoreduction of blood product before transfusion into a patient is considered the most significant recent improvement in safety and purity of blood transfusion. Leukoreduction is the process of removing >99.9% of the white blood cells (WBC) from cellular blood components (red cells and platelets).
The FDA has announced publicly that it will require that all cellular blood components transfused in the U.S be leukoreduced by the year 2002. Worldwide, ten countries, including Canada, Britain, France, Portugal, and Germany, have mandated universal leukocyte reduction, and 13 more, including Denmark, Italy, Japan, and New Zealand, are moving toward the practice.
As in any essential step of blood processing, the step of leukoreduction is subject to quality control. In order to label a component as leukocyte-reduced (leukoreduced), the American Association of Blood Bank Standards (19th ed) requires that the residual leukocyte content in the component is <5×106 WBC/unit blood. European guidelines define leukocyte reduction as <1×106 leukocytes/unit.
FDA guidelines state that quality control testing of leukocyte-reduced units should be performed on at least 1% of products (or 4/month for facilities preparing <400 units/month) and that 100% of tested units should contain <5×106 residual leukocytes/unit.
During the 1990's numerous methods were developed for testing for residual leukocytes, based on light microscopy, fluorescence labeling, or DNA amplification. In 1998, FDA approved three protocols for QC control of leukoreduced blood products:                1. Manual counting of cells in the fixed volume of transfusion product, using a Nageotte hemacytometer.        2. Flow cytometric methods, which use fluorescent beads or labeled chicken erythrocytes as internal control, and        3. The Imagn 2000 device for analyzing a fixed blood samples in a capillary chamber.        
Recent methods have limitations for proposed centralization of testing services for leukoreduced blood products in terms of sample storage time and their integrity. These methods are based on actual cell counts using cell shape, size and brightness to discriminate cell from background. Both manual and automated approaches require fresh blood, because these described parameters change with blood storage, or are sensitive to sample preparation techniques. All traditional methods are very sensitive to blood storage time because cells' deterioration and changing of size and/or shape during storage or the freezing-thawing process.
According to expert opinion, “Wide scale testing has shifted to more automated methods. However, deterioration of samples as a result of prolonged sample shipment remains an obstacle to centralized testing service.” (Walter H. Dzick, Von Sang 2000, 78, 223:226). “The requirement of fresh samples for processing is particularly problematic in connection with applications such as Quality control of WBC-reduced blood components by blood centers.” (T.-H. Lee, M. P Busch, Transfusion, 1998.38. 262:270; Transfusion, 2001, 41.276: 282.).
Other problems with such conventional methods include timing of the analysis. For example, the time required for one sample analysis using flow cytometry (sampling and analysis of histogram) is too long (3-10 min for a single sample analysis) to permit large scale testing. Further, flow cytometry and especially manual Nageotte methods both require well-trained personnel for consistent and accurate result interpretation.
Problems with traditional methods of analysis have led to implementation of DNA based analysis for quality control of WBC-reduced component has recently been recognized as an alternative of traditional “cell shape, size, and brightness” methods. Typically, DNA analysis is based on the fact that only white blood cells in human blood have genomic DNA. All other blood cellular elements, red cells and platelets are nuclei-free. The majority of all white blood cells (>99%) are not proliferating. Thus they have a known, relatively constant amount of DNA that is equal to about 7 pg per cell. Previous approaches to this technology claimed substantial, but not total, DNA recovery from whole blood samples.
One method has been evaluated with respect to quantitative recovery of WBC-associated DNA when the target cell population is present at very low concentration in test samples, and this method uses the Polymerase Chain Reaction (PCR) to detect and quantify genetic material in frozen whole blood samples. However, a Quantitative PCR protocol takes many hours to process. That is even slower in some ways than flow cytometry.
Further, PCR methods are very expensive. They require unique real time PCR devices and expensive PCR reagent kits. The preparation of calibration standard samples is labor consuming and very sophisticated. The required operator's skills are at the Ph.D. level. Thus there is a clear need for a simple, effective, and rapid method for analyzing blood transfusion products for leukocyte or other nucleotide-containing contaminants.
It is desirable to adapt the present technology and modify it for specific use such as for forensic art or for detection of leukocytes in blood products, including leukoreduced blood. Additionally, it would be advantageous to be able to rapidly qualify and quantify nucleic acid on media, either in correlation with such uses, or independent thereof.